Use of isolated domains of type IV collagen to modify cell and tissue interactions

ABSTRACT

The instant invention demonstrates that the 7S domain of type IV collagen disrupts cell aggregation and tissue development. Structural changes in mesoglea, inhibition of cell proliferation, and changes in cell differentiation patterns accompanies the blockage of cell aggregates which indicate that blockage may be due to alterations in mesoglea (extracellular matrix) structure with accompanying effects on cell behavior. Type IV collagen has a critical role in the initial formation of mesoglea and that perturbation of mesoglea formation affects cell division, cell differentiation, and morphogenesis.

This application is a continuation of U.S. application Ser. No.09/723,791 filed Nov. 28, 2000, now U.S. Pat. No. 6,448,222, which is acontinuation of U.S. patent application Ser. No. 09/183,548 filed Oct.30, 1998, now U.S. Pat. No. 6,384,012, which is a continuation of U.S.application Ser. No. 08/800,965 filed Feb. 18, 1997, now U.S. Pat. No.5,856,184, and a continuation of U.S. patent application Ser. No.08/497,206 filed Jun. 30, 1995, now U.S. Pat. No. 5,691,182, which is acontinuation of U.S. application Ser. No. 08/268,969 filed on Jun. 30,1994, now U.S. Pat. No. 5,567,609.

STATEMENT OF RIGHTS

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grants No.01-RR06500 and AM 18381 awarded by the National Institute of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for the manipulation of intercellularand intertissue interactions. The instant invention provides methods forthe inhibition of cell adhesion to extracellular matrix components orthe formation of functional basal lamina, and the manipulation of theresults of such attachments. Thus the instant invention to themodification of cellular interactions, with extracellular components,and methods for maintaining cell phenotype, developmental stage, andplasticity in vivo and in vitro.

2. Description of the Prior Art

The basement membrane (basal lamina) is a sheet-like extracellularmatrix which is a basic component of all tissues. The basal laminaprovides for the compartmentalization of tissues, and acts as a filterfor substances traveling between tissue compartments. Typically, thebasal lamina is found closely associated with an epithelium, orendothelium in all tissues of an animal including blood vessels andcapillaries. The basal lamina components are secreted by cells, and thenself assemble to form an intricate extracellular network. The formationof a biologically active basal lamina is important to the developmentand differentiation of the associated cells.

The Cnidarian, Hydra, is a simplified metazoan whose body wall iscomposed of an epithelial bilayer with an intervening extracellularmatrix (ECM) termed the mesoglea. Hydra mesoglea have been shown to havea number of components seen in the ECM or basement membranes of higherinvertebrates and vertebrates, these include: fibronectin, type IVcollagen, laminin, and heparan sulfate proteoglycan. Hydra cellaggregation involves the complete morphogenesis of adult hydra frompellets of dissociated hydra cells. During this developmental process,cells segregate into an epithelial bilayer and then deposit a newextracellular matrix prior to the continuation of morphogenesis.

Extracellular matrix (ECM) components play a critical role indevelopment through their affects on such cell processes as celldivision, cell attachment, cell migration, and cell differentiation(reviewed by Timpl et al., 1989, Int. Rev. Exp. Pathol. 29:1-112; Damskyand Bernfield. 1991, Current Opn. in Cell Bio. 3:777-778; Hynes, 1992,Cell 69:11-25). It has been established that ECM/cell interactions areutilized by a wide range of vertebrate and invertebrate species toinclude such primitive organisms as the Cnidarian, Hydra. Hydra isparticularly interesting in this regard because it represents one of thefirst animal phyla to develop defined tissue layers separated by anacellular extracellular matrix (Field et al., 1988, Science239:748-752). Previous studies have shown that hydra ECM, termedmesoglea, contains type IV collagen, laminin, fibronectin, and heparansulfate proteoglycans (Sarras et al., 1991a, Dev. Biol. 148:481-494).These molecules are continuously synthesized and deposited into themesoglea in adult hydra and during hydra head regeneration (Hausman etal., 1971. J. Exp. Zool. 177:435-446). Other studies have shown thatdevelopmental processes in hydra such as head regeneration are dependenton the normal formation of ECM. These studies have shown that headregeneration in hydra morphogenesis can be blocked by using drugs thatperturb collagen cross linking or drugs that interfere with proteoglycanGAG chain extension (Sarras et al., 1991b, Dev. Biol. 148:495-500).These studies have most recently been extended to the hydra cellaggregate system. This system allows one to form a pellet withdissociated hydra cells and then observe the complete regeneration ofthe adult hydra body within 72-96 hours though the process ofcytodifferentiation and morphogenesis (Gierer et al., 1972, Nature NewBiol. 239:98-101; Sarras et a, 1993, Dev. Biol. 157:383-398). Suchstudies of hydra development and the role of the ECM have focusedheavily on a chemical approach (Barzanski et al., 1974, Amer. Zool.14:575-581; Sarras et al., 1991ab, supra). Hydra cell aggregates firstform an epithelial bilayer and then deposit an ECM before morphogenesisproceeds. Hydra cell aggregate development is blocked by drugs thatperturb ECM formation and by antibodies raised against isolated hydramesoglea. These studies demonstrate that functional studies of ECM/cellinteraction can be carried out under in vivo conditions with hydra.

Type IV collagen has been shown to be a major structural component ofbasement membranes and has also been shown to be present in hydra ECM.The protomeric form of type IV collagen is formed as a heterotrimer madeup from a number of different subunit chains called α1(IV), α2(IV) etc.The type IV collagen heterotrimer is characterized by three distinctstructural domains: the non-collagenous (NC1) domain at the carboxylterminus: the triple helical collagenous domain in the middle region;and the 7S collagenous domain at the amino terminus (FIG. 1) (Martin etal., 1988, Adv. Protein Chem. 39:1-50; Gunwar et al., 1991, J. Biol.Chem. 266:14088-14094). Type IV collagen exists as a supramolecularstructure in ECM and this structure is thought to serve as a frameworkwhich provides mechanical stability to ECM (Timpl et al., 1986, supra)and as a scaffolding for the binding and alignment of other ECMmolecules such as fibronectin, laminin, eutectin, and heparan sulfateproteoglycans (Gunwar et al., 1991, supra). The biological function oftype IV collagen is critically related to the formation of an intact ECMsince disruption of collagen cross linking by β-aminopropionitrileinterferes with the mesoglea formation and this leads to a blockage innormal hydra morphogenesis (Sarras et al., 1991b, 1993, supra).

Hydra cell aggregate development involves complete morphogenesis ofadult hydra structures within 96 hr from pellets formed with dissociatedhydra cells (Grierer et al., 1972, supra; Sato et al., 1992, Dev. Biol151:111-116; Technau et al., 1992, Dev. Biol. 151:117-127: Sarras etal., 1993, supra). Morphologically, hydra cell aggregate development canbe divided into two stages. The initial stage is from Time 0 to 24 hrwhen aggregates develop from a solid cell mass into a fluid-filled cystwhere the outer wall is formed from an epithelial bilayer with anintervening ECM termed mesoglea. This stage involves active cell sortingbetween ectodermal and endodermal cells (Technau et al., 1992, supra)and subsequent mesoglea formation once the bilayer is established. Thelater developmental stages (24-96 hr) involve processes normallyassociated with tissue histogenesis; namely, alterations in the shape ofepithelial layers, cell migration, cell differentiation, and otherprocesses that result in morphogenesis of foot, head, and tentaclestructures. In regard to the initial stages of hydra cell aggregatedevelopment, it has been shown that head regeneration in aggregates isnot due to the clustering of cells from the original head regions. Ithas been suggested that head regeneration arises de novo (Gierer et al.,1972, supra; Technau et al., 1992, supra) from foci of developmentgradients established around the spherical aggregate. This indicatesthat cell differentiation or transdifferentiation into head region cellsactively occurs during hydra cell aggregate development. In addition topositional information and possible activator influences, cells maydifferentiate or transdifferentiate under the influences of otherdevelopmental cues such as signals arising from the ECM.

Previous studies have shown that in vertebrates, fibronectin interactswith various collagens during matrix assembly, including type IVcollagen (Carter, 1984, J. Cell Bio. 99:105-114). In addition,antibodies to the collagen binding domain of fibronectin had the abilityto block ECM assembly by human lung fibroblasts. (McDonald, 1982, J.Cell Biol. 92:485-492). Other studies raised doubts as to theinteraction, while polyclonal antibodies to the collagen binding domainblocked matrix assembly, purified collagen binding domains had noinhibitory effects in this assembly process (McDonald et al., 1987, J.Biol. Chem. 262:2957-2967; Hynes, 1990, Cell 48:549-554). In generalfibronectin (FN) appears before collagen during assembly of vertebratematrices, however, in the case of hydra ECM formation, FN and collagenappear in the mesoglea about the same time, based on immunofluorescentstudies. Type IV collagen has been implicated as important in severalhuman diseases (Hudson et al., 1993, J. Biol. Chem. 268:26033-26036).Basement membrane and its components have a role in lymphocyte adhesion,migration and proliferation (Li and Cheung, 1992, J. of Immunology149:3174-3181).

The fundamental role ECM plays in tissue development and celldifferentiation reverberates across phyla and kingdoms, to focusattention on the most basic elements that are required for all tissueinteractions. The use of hydra as a model system for the study of basicelements of complex tissue interactions is a recognized approach.Instead of attempting to deduce the interaction between isolated tissuesof higher order animals, the same mechanisms and phenomenon can beexamined in vivo by using the complete animal, in hydra. This approachhas led to the use of hydra to study the effects of glucose on tissuemorphology, in an effort to understand the pathological effects ofuncontrolled diabetes on kidney glomeruli, with excellent results (Zhanget al., 1990, Diabetologia 33:704-707).

Recently the β1-laminin gene has been cloned and sequenced in hydra,showing very high homology with the human counter part. The homologuesof fibronectin and collagen are present as well. It is a reflection onthe fundamental role ECM plays, that hydra and higher order animals showthe same cell matrix interactions, with similar components, domaininteractions, receptor molecules and response to extracellular signals.Mammalian and even human hormones, when applied to hydra result inbioactivity and effect on cell behavior. It is possible to use humaninsulin to stimulate cell proliferation in hydra. Other such cross-phylaactivities can be attributed to many growth factors as well, i.e. EGF(epidermal growth factor), TGF-β, FGF (fibroblast growth factor), PDGF,to name a few.

Specific methods for the manipulation of cell adhesion to ECM, basallamina, or adjacent cells would be useful for the in vivo manipulationof tissues and cells. Methods which address the fundamental elements ofbasic cell and tissue interactions are applicable to all systems whichexhibit similar characteristic features. Such in vivo uses include, andare not limited to, inhibition of basal lamina formation, inhibition ofbasal lamina/cell interactions, and to encourage cells to maintainphenotypic plasticity. Such methods will also be useful for the in vitromanipulation of cells and tissues, for instance in maintaining cellcultures in undifferentiated or homeostatic states, non-enzymaticdispersal of cells from attachments, or the maintenance of confluentcells in suspension for propagation, maintenance, or collection.

SUMMARY OF THE INVENTION

The instant invention provides methods for inhibiting basal laminamembrane formation, in cell or tissue development, comprising contactingthe cell or tissue with one or more isolated domains of type IVcollagen. The instant invention also provides methods for in vitrocultivation of cells comprising contacting the cells to be cultivatedwith an isolated domain of type IV collagen to disrupt the formation ofbasal lamina or extracellular matrix contacts. The instant inventionfurther provides methods for disrupting basal lamina membrane formationin tissues comprising contacting the tissues with an isolated domain oftype IV collagen. In a specific embodiment of the instant invention, theisolated domain of type IV collages is the 7S or NC1 domain, or proteinconstructs having substantially the same structure as the activeelements within the 7S or NC1 domain.

Thus the instant invention provides methods for the interference withcell interactions with basal lamina components which comprisescontacting the cells or tissues with an isolated domain of type IVcollagen, and in a preferred embodiment the isolated domain is eitherthe 7S domain or the NC1 domain of type IV collagen or substantiallyhomologous protein constructs thereof which contain the specificstructural elements within the 7S and NC1 domain that convey activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are two diagrams illustrating segments of type IV collagen (1A)and fibronectin proteolytic fragments (1B) used in blocking experiments.

FIG. 2 is a graph which illustrates results of the blockage of aggregatemorphogenesis observed with NC1 domain monomer and 7S domain, when alltype IV collagen fragments are compared on an equal molar basis (0.03μM). From left to right: control group; NC1 monomer, NC1 hexamer, 8-kDafragment of NC1; 7S domain; type I collagen. Results are reported as %of normal morphogenesis as described below.

FIG. 3 are photographs of representative Hydra cell aggregates at 96 hrof development. Control aggregates develop head and tentacle structures(3A) while blocked aggregates remain in the characteristic 24-hr cysticstage (3B). Bar=286 μm.

FIG. 4 is a graph which illustrates the concentration effect of thevarious blocking agents. At higher concentrations, NC1 domain hexameralso showed blocking effect, whereas no blockage was observed with the80-kDa fragment of NC1. From left to right: control group; NC1 monomer(MN) at 0.03 μM; NC1 domain hexamer (HEX) at 0.1 μM; NC1 domain hexamer(HEX) at 0.03 μM; NC1 domain 80-kDa fragment (80K) at 0.3 μM; NC1 domain80-kDa fragment (80K) at 0.03 μM; 7S domain (7 S) at 0.1 μM; and 7Sdomain (7S) at 0.03 μM. Results are reported as % of normalmorphogenesis as described below.

FIG. 5 are electron micrographs showing representative morphology ofcontrol (5A) and blocked (5B) aggregate cells.

FIG. 6 is a graph which illustrates the results of the blockage ofaggregate formation using fibronectin and fibronectin derivedproteolytic fragments. All samples were tested at 0.5 mg/ml. From leftto right: BSA (Bovine Serum Albumin); FN (intact fibronectin); 120K (the120-kDa fragment of FN); 45K (the 45-kDa fragment of FN); 40K (the40-kDa fragment of FN); 30K (the 30-kDa fragment of FN); RGDS (RGDSpeptide); RADS (RADS peptide). Results are reported as % of normalmorphogenesis as described below. The effectiveness of the blockingagents was found to be concentration dependent.

FIG. 7 illustrates the effects of NC1 (Hexamer) and 7S domains of TypeIV collagen at a 50 ug/ml concentration on angiogenesis from mousethoracic aorta organ cultures.

FIG. 8 illustrates the effects of 7S domain of Type IV collagen onangiogenesis from mouse thoracic aorta organ cultures. The domainconcentrations employed in this experiment were 0 ug/ml (control); 0.5ug/ml; 5 ug/ml and 50 ug/ml.

FIG. 9 illustrates the effects of NC1 (Hexamer) domain of Type IVcollagen on angiogenesis from mouse thoracic aorta organ cultures. Thedomain concentrations employed in this experiment were 0 ug/ml(control); 0.5 ug/ml; 5 ug/ml and 50 ug/ml.

FIG. 10 are photographs of mouse thoracic aorta segments embedded inMatrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture. Control specimen (0ug/ml of NC1 (Hexamer) and 7S domains) exhibited growth of microvesselsfrom the cultured tissue into the matrix (FIG. 10A). In contrast,angiogenesis was inhibited in specimens cultured with 50 ug/ml of 7Sdomain (FIG. 10B) and NC1 (Hexamer) domain (FIG. 10C).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Our studies indicate that the formation of an appropriate ECM structureinvolving fibronectin and type IV collagen is critical to cell aggregatedevelopment and that perturbation of ECM formation adversely affectscell division and cell differentiation during the formation of complexcell aggregations and tissues. This is illustrative of the critical rolethe ECM and cell-ECM interactions play in tissue development in allorganisms that have differentiated tissues. Structural changes inmesoglea, inhibition of cell proliferation, and changes in celldifferentiation patterns accompanied the blockage of cell aggregates.Thus type IV collagen is critical to the early stages of cell aggregatedevelopment when the mesoglea is initially formed and that perturbationof aggregate development by fragments of type IV collagen results inalterations in hydra cell division, cell differentiation, andmorphogenesis. It is demonstrated that type IV collagen components are acritical element in the formation of any cell to ECM (basal lamina)contacts, and that by applying the proper methods, this interaction canbe manipulated in a predictable fashion.

The following examples are meant to illustrate specific embodiments ofthe instant invention, and are in no way intended to limit the breadthor scope of the teachings embodied in the instant specification. Onewith ordinary skill in the art will be able to take the teachings of theinstant specification and use the instant invention in other specificembodiments.

EXAMPLE 1 Disruption of Cell Aggregate Development

All experiments utilized Hydra vulgarie (previously named Hydraattenuata). Animals were cultured as described previously (Sarras etal., 1991a, supra) and were not fed for 24 hr prior to use.

Hydra cell aggregates were prepared according to Gierer et al., (1972,supra) with modification described by Sans et al., (1993, supra). Theywere incubated either in microtiter plates (NUNS, Denmark) with oneaggregate/well/10 μl incubation solution or in 96-well plates (Falcon)with three to five aggregates/well/50 μl incubation solution.Antibiotics were used throughout all steps of aggregate preparation toassure that the aggregates used in this study were free of any symbionicbacterial populations (Sarra et al., 1993, supra). Various analysisindicated that aggregates were free of bacteria under these conditions.By 24 hr of cell pellet formation, hydra cell aggregates develop septatejunctions between epithelial cells (Wood et at., 1980. J. Ultrastruc.Res. 70:104-117). These junctions prevent the introduction ofmacromolecules from medium into mesoglea. Therefore, exogenous matrixprobes (e.g., fibronectin, type IV collagen, and type IV collagenfragments, peptides or antibodies) were added to the culture mediumimmediately after pellet formation (Time 0). Control hydra cellaggregates were cultured in hydra medium, bovine serum albumin (Sigma.St. Louis, Mo.), or nonimmune serum. After 24 hr of incubation,aggregates (both control and experimental groups) were transferred intofresh hydra medium and culture was continued until 96 hr.

Type IV collagen fragments used in blocking experiments are illustratedin FIG. 1 (A). The NC1 monomer—a mixture of α1(IV), α2(IV); NC1 hexamer,80-kDa protomeric truncated fragment (NC1 domain with part of triplehelix); and the 7S domain, were all obtained from enzymatic digestionand subsequent chromatographic purification of bovine kidney glomerulibasement membrane or bovine lens basement membrane (Langeveld et al.,1988, J. Biol. Chem. 263:10481-10488; Gunwar et al., 1991, supra).Antibody to the NC1 (α1(IV)+α2(IV)) domain of type IV collagen wasgenerated in rabbits immunized with monomeric subunits isolated from theglobular domain of bovine kidney basement membrane type IV collagen(Langeveld et al., 1988, supra). The precise molar concentrations ofthese fragments was determined by spectrophotometry and amino acidcomposition analysis.

Criteria for morphogenesis was as follows. The morphological developmentof hydra cell aggregates was studied using a dissecting microscope(Wild, Herbrugg). Observations were carried out at Time 0, 24, 48, 72,and 96 hr. The normal morphogenesis of hydra cell aggregates betweenTime 0 and 96 hr has been described previously by Gierer et al., (1972,supra) and Sums et al., (1993, supra). Abnormal development (blockage)of hydra cell aggregates was considered if an aggregate did not showhead and tentacle structures at 96 hr. i.e., was retained in a cysticstage (FIG. 3). The percentage of aggregates-with head and tentaclestructures at 96 hr of development was calculated and data for eachgroup from different experiments were pooled and plotted. A minimum offive aggregates were tested per group and all experiments were repeatedat least three times except where indicated. Using an ANOVA statisticaltest, a P value <0.05 was taken as the level of significant differencein all groups analyzed. The ANOVA Apical test was used for all dataanalysis.

Transmission electron microscopy was used to examine the fine structureand morphology of control and treated aggregates. Cell aggregates wereimmersed in Karnovsky's fixative overnight at 4° C. and postfixed in 1%OsO₄ for 1 hr. Samples were then stained en bloc in 0.5% uranylmagnesium acetate overnight at 4° C. After dehydration and infiltration,samples were embedded in Spurr's resin. Blocks were cut with aReichert-Jung microtome, stained with uranyl acetate and lead citrate,and viewed using a JOEL 100S transmission electron microscope.Morphometry analysis was carried out as described previously (Zhang etal., 1990, supra).

Immunofluorescent screening for type IV collagen in hydra cellaggregates at various time points after pellet formation showed thatsignal was detected at 48 hr, and became progressively stronger andremained in the mesoglea throughout all later stages of aggregatedevelopment. FIG. 3 illustrates the typical appearance of control andblocked aggregates at 96 hr. Blocked aggregates failed to develop beyondthe 24 hr stage, and either remaining in a cystic stage ordisaggregating into dissociated cells by 96 hr.

As shown in FIG. 2, the 7S domain, and monomers of NC1 domain were mosteffective in blocking hydra cell aggregate morphogenesis when fragmentswere tested on an equal molar basis. This blockage was alsoconcentration dependent. NC1 hexamer blocked aggregate development athigher concentrations whereas the 80-kDa fragment showed no effect (FIG.4). Antibody to NC1 domain had a similar blocking effect (Table 1).

TABLE 1 Treatment with Antibody to ECM Components Treatment % of normalmorhogenesis nonimmune serum 1:10-1:100 79 ± 7.25 (54/68)^(a) Anti-NCl1:10^(b) 0 (0/15) Anti-NCl 1:40 0 (0/6) Anti-NCl 1:80 100 (3/3) Anti-FN1:10^(c) 0 (0/15) Antu-FN 1:50 0 (0/12) Anti-FN 1:100 32 ± 15.5 (10/30)Anti-FN 1:200 33 ± 19.25 (5/15) ^(a)Data are presented as means ± SEM(No. with normal morphogenesis/total No. treated). ^(b)Polyclonal Abagainst NCl (α1(IV) + α2(IV)) domain of bovine kidney type IV collagen.^(c)Polyclonal Ab (ICN Biochemicals) against human plasma fibronectin.

Similar studies were carried out using fibronectin as the inhibitor.Intact fibronectin, proteolytic fragments, RGDS peptides, and antibodyto fibronectin were tested to determine their effect on hydra cellaggregate development. FIG. 1(B) shows a diagram of the relationship ofthe various fragments to the intact fibronectin molecule. Of these,intact fibronectin and its 30-KDa gelatin binding domain were found tobe effective at 0.5 mg/ml in blocking aggregate development (FIG. 6).Effectiveness was found to be concentration dependent. Antibodiesagainst fibronectin also showed blocking effects (Table 1).

Transmission electron microscopy was used to study the ultrastructure ofmesoglea under the influences of each exogenously introduced ECMmolecule. Blocked aggregates were processed for TEM analysis at varioustime points of experiments and the ultrastructure of their mesoglea wascompared to that of control aggregates fixed at the same time ofdevelopment. As compared to control group specimens (FIG. 5A), theultrastructure of mesoglea in blocked aggregates was reduced inthickness, irregular at its epithelial border, and appeared to have lostsome of its normal ultrastructural organization (FIG. 5B). Morphometricanalysis (Table 2) indicated that the mesoglea of blocked aggregates wasreduced in thickness by approximately 50% compared to controls and thisreduction was significant as determined using statistical analyses. Thisaltered mesoglea ultrastructure was observed with all blocking reagentstested.

TABLE 2 Morphometric Analysis Interepithelial width of mesoglea asTreatment (from Time 0 to 24 hr) reflected by area measurements^(a)BSA^(b) 1434 ± 582  30 kDa^(c) 724 ± 245* Anti-FN^(d) 969 ± 458  ^(a)AllEM micrographs of mesoglea were taken at 8,000 X magnification andprinted at the same size. An 80-mm section was bracketed along mesogleaon each print and mesoglea within the bracketed area was measured bycomputer progam, SigmaScan. The measuring unit is mm². Under thesemeasuring conditions, area reflects interepithelial width changes aspreviously described (Zhang et al., 1990, supra). Data are representedas mean ± SD. ^(b)Bovine serum albumin at 0.1 mg/ml concentration.^(c)Fibronectin 30-kDa gelatin binding fragment at 0.1 mg/ml.^(d)Antibody to fibronectin at 1:100 dilution. *Statistically differentfrom BSA group (P value <0.05).

To determine if inhibition of cell proliferation was responsible for theblockage of cell aggregate morphogenesis, aggregates were treated fromTime 0 to 96 hr with 10 μM hydroxyurea (HU), which has been shown toinhibit DNA synthesis in hydra cells (Bode et al., 1976, J. Cell Sci.20:29-46). Although HU completely blocked DNA synthesis, the drug didnot inhibit morphogenesis of hydra cell aggregates.

A critical role for collagen in the process of matrix formation followsfrom previous pharmacological studies involving lanthrytic agents(Barzanski et al., 1974, supra; Sarras et al., 1993, supra). In thepresent demonstration, type IV collagen domains were effective inperturbing mesoglea formation and in blocking aggregate development. Inthis regard, the NC1 domain monomer and the 7S domain fragment were mosteffective in blocking aggregate development when all type IV collagenfragments were compared on an equal molar basis. Others have proposedthat the NC1 domain and the 7S domain are the sites at which the type IVcollagen protomer is involved in intermolecular ridging during formationof the supramolecular network (Martin et al., 1988, Adv. Protein Chem.39:1-50).

It is not clear why the larger NC1 hexamer and the 80-kDa fragment wereless effective than the smaller NC1 monomer in blocking development. Thesame phenomenon was seen with fibronectin, while the most effectiveblocker, the 7S fragment, has a mass greater than 100 kDa. On a totalmass basis, the 7S domain has a relatively high proportion ofcarbohydrate residues associated with the polypeptide chain (15-18%),and further analysis of these carbohydrate residues indicate that theyhave unique structural features such as the presence of terminal α-D-Galresidues on N-linked oligosaccharide groups (Langenveld et al., 1991,supra; Nayak et al., 1991, J. Biol. Chem. 266:13978-13987). Theinability of type I collagen to block development is consistent with thefact that it has not been detected in hydra mesoglea.

The effect of mesoglea components on hydra cell behavior may be viewedat two levels. At one level, mesoglea may be viewed as simply astructural entity which is required as a foundation for stability andmaintenance of the epithelial bilayer. At another level, however, ECMcomponents in mesoglea provide developmental cues which nodule such cellprocesses as cell division, migration, and differentiation during hydramorphogenesis. It is clear that ECM components are not simply structuralmolecules. Fibronectin and type IV collagen have been shown to controlendothelial cell proliferation and differentiation (Tagami et al., 1992,Cell Tissue. Res. 268:225-232; Ingber, 1990, PNAS USA 87:3579-3583). Ithas been shown that the mechanochemical interactions between striatedmuscle cells in jellyfish and grafted mesoglea can induce or inhibit DNAreplication and cell transdifferentiation. In the present examples,while cell proliferation is inhibited in morphologically blockedaggregates, it is also apparent that normal aggregate morphogenesis canoccur even in the absence of cell proliferation. Therefore, a reductionof cell division in blocked aggregates can not account for the blockageobserved in aggregate morphogenesis.

The homology between the hydra model system and what is known about thedevelopmental and regulatory mechanisms of higher organisms makes theuse of type IV collagen fragments in this system applicable to the samecell and tissue interactions in other organisms. The instant methods areapplicable to, among other things, the inhibition of metastasis, controlof cell division, reduction of scar tissue formation, intervention inepithelial tissue formation, inhibition of angiogenesis, reduction ofcomplications due to cell adhesion in organ transplants, inhibition ofangiogenic invasion of tissue, or the inhibition of lymphocyte adhesionand mobility.

EXAMPLE 2 Cell Culture Maintenance

The methods of the instant invention are quite useful for themaintenance of cells in culture, where such maintenance requiresmaintenance of cell phenotype and morphology with minimal adhesion. Itis well recognized that there are many critical factors that contributeto the successful maintenance and propagation of cells in culture.Namely cell division can be anchorage dependent, and in some instanceswill show density-dependent inhibition. Generally most cultures madefrom cells dissociated from tissues, unlike bacteria, are not adapted toliving in suspension and require a solid surface on which to grow anddivide. Cells are now grown in cultures, so that they adhere to plastic.Cells can vary in the requirements of culture media, and the nature ofthe supports, and some cells will not grow without the proper ECMcomponents coated on the plastic dish. Recognized methods for mammaliancell culture can be found in such general references as “Mammalian CellBiotechnology,” edited by M. Butler, Oxford University Press. 1991, and“Readings in Mammalian Cell Culture.” 2nd Edition, edited by R. Pollack,Cold Spring Harbor Laboratory, 1981.

The methods of the instant invention will provide a means by which therequirement for adhesion is eliminated by providing the cells with aneffective amount of the type IV collagen domains which will allow thecells to remain in solution while still appearing to be bound to ECM.This will greatly improve the use of cell cultures in bioreactors andother large scale commercial applications. The use of NC1 or 7 S domainin about the same effective concentration per cell as demonstrated inthe previous example will keep cultures of cells effectively boundwithout adhesion. Further, isolation and manipulation of cells will notrequire the use of general a pharmacological agents which effect manycellular function in an imprecise and non-specific manner.

Primary cultures, isolated directly from animal tissues, can be used toform secondary cultures of specific cells. In this manner, the cells canbe subcultured for several weeks or months, displaying the phenotype andmorphology of the parent cells. Most vertebrate cells will die inculture after a finite number of divisions, for example human skincell's can typically last for several months dividing from 50 to 100times before they die. Variant cell lines can arise however, which areimmortal in that they can be propagated in cell culture indefinitely.These cells usually grow best when attached to a solid support, andtypically will cease to grow once they have formed a confluent layer onthe surface, demonstrating contact inhibition. Cell lines prepared fromcancer cells differ from those prepared from normal cells in many ways.Such cells tend to proliferate without solid support and will grow tomuch greater density than normal cells.

Thus the methods of the instant invention can be used to affect primaryand secondary cell cultures so that they behave as if in contact with asolid support, but without the effects of cellular contact inhibition.Thus parent cell lines can be maintained in vitro under conditions thatwill promote the maintenance of cell phenotype and morphology.

One area of great interest is the isolation and culture of embryonic orpluripotent stem cells in vitro for eventual manipulation and use invivo. The isolation and maintenance of such relatively undifferentiatedcells, and the propagation of such cells would allow for the productionof vast amounts of specific cells which could form the basis of tissueregeneration or replacement. Thus the teachings of the instant inventioncan be applied to the maintenance of pluripotent cell isolates, andallow for the propagation of these cells while inhibiting morphologicalchanges and differentiation in vitro.

EXAMPLE 3 In Vitro Effect on Angiogenesis

Angiogenesis, the process of formation of new blood vessels, plays animportant role in physiological processes such as embryonic andpostnatal development as well as in wound repair. Formation of bloodvessels can also be induced by pathological processes involvinginflammation (e.g., diabetic retinopathy and arthritis) or neoplasia(e.g., cancer) (Folkman, 1985, Perspect. Biol. Med., 29, 10).Neovascularization is regulated by angiogenic growth factors secreted bytumor or normal cells as well as the composition of the extracellularmatrix and by the activity of endothelial enzymes (Nicosia andOttinetti, 1990, Lab. Invest., 63, 115).

During the initial stages of angiogenesis, endothelial cells sproutsappear through gaps in the basement membrane of preexisting bloodvessels (Nicosia and Ottinetti, 1990, supra; Schoefl, 1963, VirehousArch. Pathol. Anat. 337, 97-141; Ausprunk and Folkman, 1977, Microvasc.Res. 14, 53-65; Paku and Paweletz, 1991, Lab. Invest. 63, 334-346). Asnew vessels form, their basement membrane undergoes complex structuraland compositional changes which are believed to affect the angiogenicresponse (Nicosia et al., 1994, Exp. Biology, 164, 197-206). Earlyplanar culture models have shown that basement membrane moleculesmodulate the attachment, migration and proliferation and organizationalbehavior of endothelial cells (Nicosia et al., 1994, supra). More recentstudies with three-dimensional aortic culture models which more closelysimulate angiogenic conditions that occur during wound healing in vivosuggest that basement membrane is a dynamic regulator of angiogenesiswhose function varies according to its molecular components (Nicosia,1994, supra).

With modifications, the procedures of Nicosia and Ottinetti (1990),supra, and Nicosia et al (1994), supra, were utilized for experimentsdesigned to test the effect of Type IV collagen on angiogenesis under invitro conditions. The model has been used to study the effects of growthfactors and extracellular matrix molecules on the angiogenic responseand employs aortic rings cultured in three-dimensional collagen gelsunder serum-free conditions. These experiments are outlined below.

A. Methods

Experiments were performed with Swiss Webster male mice which were 1 to3 months old. Following anesthesia, the thoracic aorta was excised underaseptic conditions and transferred to sterile MCDB 131 sterile growthmedium (Clonctics, San Diego, Calif.) containing antibiotics. Fat wasdissected away from the aorta and approximately six to eight 1 mmthoracic segments were obtained from each specimen. Segments weretransferred to 48 well tissue culture plates. The wells of these plateswere layered with 100 microliters of Matrigel (EHS basement membrane,Collaborative Biomedical Products, Bedford, Mass.) prior to transfer ofthe aortic segments. The Matrigel was diluted 1:1 with MCDB 131 growthmedium prior to use. The segments were centered in the wells and anadditional 100 microliters of Matrigel was then placed over thespecimens. The aortic segments were therefore embedded in the basementmembrane matrix. Each well then received 300 microliters of MCDB 131growth medium. The plates were placed in an incubator maintained at 37°C. with 5% CO₂. Specimens were observed daily over a 7 day period. Newlygrowing microvessels were counted using an inverted phase microscope atvarious times during the culture period, but data is expressed at 3 and5 days of culture. To test for the effect of Type IV collagen onangiogenesis, domains at known concentrations are mixed with theMatrigel and with the MCDB 131 growth medium. Fresh MCDB 131 growthmedium (plus and minus collagen domains) was changed every 3 days.

B. Results

After establishing the time course of angiogenesis under controlconditions (Matrigel plus MCDB 131 growth medium), experiments wereperformed using various concentrations of Type IV collagen (isolatedfrom bovine lens) NC1 (hexamer) and 7S domains. Data represents theanalysis of at least 3 specimens per experimental condition. In thefirst experiment (FIG. 7), analysis indicated that a concentration of 50micrograms/ml, NC1 domain and 7S domain significantly inhibitedangiogenesis as monitored at 3 and 5 days of culture. In the secondexperiment, various concentrations of these domains were analyzed. Asindicated in FIG. 8, 7S domain at 50 micrograms/ml again significantlyinhibited angiogenesis at 3 and 5 days. Inhibition was reduced at 5 and0.5 micrograms/ml concentrations. As indicated in FIG. 9, NC1 domain wasless effective in blocking angiogenesis as compared to that observed inthe first experiment (FIG. 7). In addition, as compared to the 7Sdomain, there was less of a correlation between concentration andinhibitory action.

FIG. 10 are photographs of mouse thoracic aorta segments embedded inMatrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture in the presence orabsence of 50 ug/ml of Type IV collagen domains. The control specimen(no domains) exhibited growth of microvessels from the cultured tissueinto the matrix (FIG. 10A). In contrast, angiogenesis inhibition wasobserved in tissues cultured in the presence of 50 ug/ml of 7S domain(FIG. 10B) and NC1 (Hexamer) domain (FIG. 10C).

The results of these experiments suggest that as observed previouslywith Hydra cell aggregates, the 7S domain of Type IV collagen is moreeffective as a blocking agent as compared to the NC1 domain.

In summary, the present invention is broadly applicable to a variety ofin vivo and in vitro uses which include inhibition of metastasis,control of cell division, reduction of scar tissue formation,intervention in epithelial tissue formation, inhibition of angiogenesis,reduction of complications due to cell adhesion in organ transplants,inhibition of angiogenic invasion of tissue, the inhibition oflymphocyte adhesion and mobility, and the maintenance of pluripotentcell isolates in vitro while inhibiting morphological changes anddifferentiation.

In practicing the invention, the amount or dosage range of NC1 (Hexamer)and 7S domains of Type IV collagen employed is one that effectivelyinhibits or disrupts cell adhesion to extracellular matrix components orthe formation of functional basal lamina. An inhibiting amount of TypeIV collagen domain that can be employed ranges generally between about0.1 and about 500 ug/ml, preferably ranging between about 0.5 and about50 ug/ml.

While the fundamental novel features of the invention has been shown anddescribed, it will be understood that various omissions, substitutionsand changes in the form and details illustrated may be made by thoseskilled in the art without departing from the spirit of the invention.It is the intention, therefore, to be limited only as indicated by thescope of the following claims.

What we claim is:
 1. A method for inhibiting tumor growth comprising contacting said tumor with one or more compound selected from the group consisting of an isolated type IV collagen α2 NC1 chain domain, and an isolated antibody against a type IV collagen α2 NC1 chain domain.
 2. A method for inhibiting tumor metastasis in tissue comprising contacting said tumor or tissue with one or more compound selected from the group consisting of an isolated type IV collagen α2 NC1 chain domain, and an isolated antibody against a type IV collagen α2 NC1 chain domain.
 3. The method of claim 1 wherein the compound is the type IV collagen α2 NC1 chain domain.
 4. The method of claim 2 wherein the compound is the type IV collagen α2 NC1 chain domain.
 5. The method of claim 1 wherein the compound is the isolated antibody against a type IV collagen α2 NC1 chain domain.
 6. The method of claim 2 wherein the compound is the isolated antibody against a type IV collagen α2 NC1 chain domain. 